Anti-anemia material and method of making the same



Patented Mar; 3, 1936 UNITED STATES PATENT OFFICE ANTI-A EMIA MATERIAL METHOD MAKING THE SAME William S. McEllroy and William F. Herron,

Pittsburgh, Pa.

' No Drawing. Application June so, 1932, Serial No.'620,301

This invention relates tomethods of treating glands and tissue to produce highly concentrated,

potent preparations for use in the treatment of disease. Specifically, thisinvention relates to a method of treating animal material, such as fresh callypotent material potential in fresh liver, but

heretofore unavailable as a liver derivative.

Five years ago or more, Minot observed that the taking of raw, or partially cooked, whole liver,

daily by persons suffering from pernicious a e-- mia, produced remission of the disease, and that improvement took place aboutthe tenth day. In order to produce remission, the patient had to consume a minimum of approximately three thousand grams, or roughly, 6.6 pounds of raw or partially cooked liven. After remission, the,

7 patient was required to take, daily, approximate- 1y pound, more or less, of liver, depending upon the condition of the patient, in order to maintain the patient in'a healthy condition. Such a sustained diet of liver was, in most cases, im-

possible to maintain because the patient could;

not retain it in his stomach. Because of this revulsion of the body to a. sustained liver diet; concentrated potent preparations of liver'weredeveloped.

. Some of these preparations w'ereadapted to! be taken bymouth and others-for'intramuscular injection. These highly concentrated forms of liver overcame, to a large extent, the objection of a sustained .dail diet of raw or partially cooked' liver. vIt has been observed that fresh'liver,.for example calfs liver, containsa relatively 'small amount of preformed materials that"are bene-'.

hole] in e treatment of pernieious anemia. If raw or partially cooked liver is taken-by a patient suffering from this disease, these preformed materials produce remission,'as indicated by increase of the reticulocytes of the patients blood. Since it was found that a patient-could not endure a sustained 'diet of raw or partially cooked liver,

methods were developed whereby concentra. ed potent preparations ofliver could be produi ed having the preformed materials therein. Some of these preparations were made in powdered form and others in extract or liquid form.

Since c'alt's liver containsmore of these pre- 10 Claims. 167-74) these potent preparations very expensive, also.

j The most generally utilized method of producingtheve mentioned potentpreparations of liver, is the one devised by Minot and Cohn.

,Under this method, the preformed materials are 10 derived by a chemical process involving a series of steps depending ondifierential solubilities of a series of solvents, the last step producing what is known as fraction G which is soluble in water but insoluble in 95% alcohol. Fraction G contains only the preformed active materials, present in liver, in concentrated and potent form which produces remission in the treatment of pernicious anemia. By these methods, only the pr formed potent materials are made available fo ,thera peutic action, and such available preformed materials are only a nominal percentum of them tential supply thereof. k I In the light of our researches and observatio'ns,

.we have discovered-that the'largestproportion of the potent materials in liver which are useful and beneficial in the treatment of pemicious' anemia, are bound up in an unavailable form. This large potential supply of beneficial materials is unavailable tothe patient, if liver is taken inraw or partially cooked form, because the patients digestive system cannot liberate or extract them. w V

"This large potential supply of beneficial material has also been unavailable in the concentrated forms because the methods heretofore used for preparing concentrated and potent products of liverwill not release'or yield any of these bound up materials from their potential 40 source ofsupply. o

- At the present time, theidentity and the quan tity of the therapeutically active'materials present in these potent preparations cannot be determined by chemical analysis, because', appar ently, these. active, beneficial materials are dei stroyed in the process of analysis. Therefore, the only method, now known, of determining ,the

amount of these potent,--benefleial and therapeutically active materials present in a given An object at our invention is therefore to provide 'a methodwhereby this large potential supply of therapeutically active materials-may be provision 'ofa m'ethod of producingghighly potent released from glands and tissue andbe converted into therapeutically potent material and, thus made available for-the treatment of disease.

Another object of our invention is the provision of a method of treating liver or tissue so that the 1 it possible toobtain from calfs liverby 'methods ofthe prior art.

A still further object of ourinvention is the preparationsfidrthe treatment of diseasesuch as pernicious" shall be inexpensive, within thieglmeans of'therich andthe poor; .and so inexpensiverthat clinics may dispense the preparatioh to charity patients without subjecting themselvesjto huge expenditures of money.

Whilethe of-my inventionmaybe utilized" for the of increasing the therapeutically potent-materials obtained "from animal material. such as glands, tissues, etc., the disclosureofthe oonstitutingapart of thisinvention is limited to thetreatment of raw liver for the purpose of increasing the available theraa predetermined amount of the same, and pass- 'ing it through a device, for example, ,a meat peutically potent material contained in liver and employed in the treatment of anemia.

Generally stated, our method consists in,taking a selected form of gland or tissue, weighing out grinder to finely divide or mince the same, Such finely divided tissi'ieor'gland is placed in a container having therein an antiseptic in solution withwater and anacid. Instead of acid, we may a favorable'medium for autolysis.

.use any chemical which will produce a favorable autolytic reaction in respect to the hydrogen ion -concentration, or'any chemicals which produce The container-is then placed in an incubator, the temperature of which is maintained at apr proximately body temperature so that autolysis or enzyme action may take place. The antiseptic is added in order to prevent putrefaction ofthetissueortheglandandtheacidorother chemical is employed to give a (pH) that will be conducive toactive autolysis, enzyme :action, or

'in other words self digestion.

During the process of autolysis, the'aforemen izloned potential supplyof heretofore unavailable potent materials are rendered available. The

longer the autolysis is permitted to' go on the more thesematerialsare form able creased.

In the light of prior scient c knowledge we have drawn the conclusion ith'atthe preformed products which arepresent and-available in beef liver, and available in larger quantitiesin with liver, arethe end products of "enzyme action. Therefore, if autolysis or enzyme action is permitted to go on under such conditions that putrefactionwill not talre place, the end products availfor action may be greatly .in-

After mixture is wncentratcd by distillation; prefers ably under reduced pressure, so asto 'drive ofli autolysis is complete'the resultant the antiseptic and the water. The undistilled residue may be filtered or not, as desired. In case the residue is filtered, the residue of the illtration, i. e., the undigested materials are discarded. To the undistilled residue, whether filtered or not, a preservative, such as alcohol may be added. If it is desired to make the preparation palatable, flavoring, such as peppermint,- I

may be added.

In the treatment of liver by our method, we

take fresh liver, beef liver being preferred since it is inexpensive, and grind it in a meat grinder to finely divide the same. This ground liver is then placed in a container having water, hydrochloric acid and chloroform therein, in the proportions of about 200 grams of liver, approximately 5 c. c. of chloroform, 2 c. c. concentrated hydrochloric acid, and 1000 c. c. of water. These proportions of hydrochloric ,acid and water give thejproper degree of acidity or (pH) which is conducive to active autolysis. The chloroform is added as a preservative which permits the autoiysis to go ,on but prevents putrefaction of the liver.,-.' The. (pH) of the solution which is favorable to active autoly'sismay vary from pH 2 to 7.5, but the mostfavorable pHis between pH from 4.5

to 7. The (pH) values favorable for active autolysis maybe found in recognized text books, for example, see Wells Chemical Pathology, 5th edition, pages 66 through 72. The extent to which any material may be autolyzed depends on the dilution of the autolyzing solution. The greater the dilution, the further the material will euteiyiie before the solution is saturated with amino-acids.

For practical purposes, such a dilution is-preferred that will require the least expense in evaporating water, to concentrate the aiitolyred 'material, for a given potency.

The container is then placed in an incubator, the temperature of which is maintained at, ap-

degree of completeness of such action may be determined by chemical analysis. The-degree of completeness may be ascertained by determining what percentage of nitrogen is present in the form of'protein and in the form of amino-acid, or end products. I 1

The material which has thus undergone autoiysis is concentrated by distillation under reduced proximately 37 C. for a period averaging from Y 10 days to two weeks. During this period, the liver is broken down by self digestion, and the pressure to drive off the chloroform and the water. It'is preferred to concentrate the preparation under. reduced pressure because thefdistillation at-atmo'spheric pressure or higher, may

raise thevtemperature of thepreparation to a point which would destroy the bcneficialelements or materials therein.

If desired, the concentrated residue mayf'be filtered to remove the undigested portions of the liver; However, it has been foimd in'practice that the unfiltered concentrate may be given to p'atientswithout impairing its therapeutic action.

To preserve the concentrate, alcohol may be added) Such a concentrate tastes. very much like beeffbroth and has to the in the treatment of llver as above described,

it will be appreciated by those skilledinart, that autoly'sis will not go onto completion because the water will become saturated with the end products'such'as, amino-acid. When sat,

a slight after-taste similar, to strong cheese. This after-taste may be avoided" by adding fiavorin'g, such as concentrate. 70 l .vents further autolysis.

uration takes place, a chemical equilibrium pre- The autolyzing material, in order to be autolyzed to completion or nearly so, maybe dialyzed so as to prevent saturation or chemical equilibrium. If autolysis and dialysis are employed, the mixture of ground or minced liver, the water, hydrochloric acid, and

the chloroform is placed in a container which is divided into two chambers separated by a-partition, in which partition there. is placed 9. diaphragm of a material, such as parchment, which permits the amino-acid, or end products, to pass from the chamber in which autolysis is taking large quantities of therapeutically potent material in form available for use and which was hereclinical features with symptoms 40 gestion of the equivalent of 500-350-800 grams tofore unavailable, and that this production of potent material is accomplished by autolysis of the'liver itself wherein the 'autolic action is carried to completion or substantial completion. Y

It :has been found that the treatment of beef liver, in the manner described abovewithout dialysis, produces a preparation having more than five times the amount of potent material than can be obtained from the equivalent amount, by weight, of'raw or partially cooked calfs liver. And this wasso even though autolysis was far from complete. 4 v

Portions of autolyzed liver preparation after partial, concentration under reduced pressure,

have been fed by mouth to previously untreated cases of pernicious anemia, showing classical dicatingcord changes, typical blood picture anda; chlorhydria. Reticulocyte responses characteristic of the treatmentof pernicious anemia with potent material varying from 10 to 16% followed the oral inof liver respectively in cases studied. 'The following, chart summarizes the finding, in one of these cases, of thereticulocyte response to autolyzed' liver.

. Reticulo- Grams Time cytes I autolyzed liver fed A comparison of the above chart the reticu;

locyte responses and the grams f autolyzer liver this invention may fed tothe patient, to the amounts of liverreported in literature necessary 'to'invqke a maximal reticulocyte response, shows that autolysis will materially increase the potency 'of liverpreparationsi While the concentrate described above is an extract in liquid form it is tobe understood that the potent materials thereof maybe removed by well lmown processes, and put up in powdered form. The preparation made inaccordance with mouth or by intramuscular injection.

The fraction Greferred to herein may also be.

' such solution has obtained from the preparation resulting from liver treated'by our method by a simple'chemical process. To obtain fraction G it is merely necessary to treat the. concentrated autolyzed preparation with absolute alcohol. The active part, which is insoluble, precipitates out.

4' In the treatment of certain kinds of tissue,

to produce therapeutically active preparations,

autolysis may be made more active by adding certain inorganic substances, such as for example, manganese chloride.

Having thus described our invention, it is to be understood that only such limitations shall be placed onthe invention as are imposed by the prior art and the appended claims.

What we claim as new and desire tosecure by Letters Patent is:

1. The method of increasing the anti-anemic properties of liver by at least 2.5 times the amount 1 of such properties contained therein, that consists inautolyzing fresh liver in a, solution at about body temperature and a pH favorable for active autolysis for such a length of time that the increase in theamino-acidcontent of the solution reaches a substantially constant level.

' 2. A method of increasing the amount of potent materials inraw liver by at least two and onehalf times as determined by the amount of raw liver required to produce maximal reticulocyte response in pernicious anemia, that consists in aritolyzing liver in an antiseptic acid solution having a pH favorable to active autolysis, and continuing such autolysis, to such a degree of completeness that the solution is substantially,

saturatecLwith amino-acids. i

3. Themethod of increasing the anti-anemic properties of liver'to at least 2.5 times the amount of such properties contained in an equalr'amount of raw liver thatconsists in autolyzing fresh liver in a solution having 'a pH favorable to active autolysis for such a length of time that such in-' crease is effected as determined by biological assay.

4. The method of increasing the anti-anemic properties of liver by at least 2.5 times that consists in autolyzing fresh liver in an antiseptic solution having a pH favorable for; active autoland'continuing such autolysis to such a degree of completeness that such increase of properties is a concentrate derived from liver autolyzed in a solution having a pH favorable for active-auto ysis andfor such a length of time that the amino-acids reach asubstantially constant level.- 6. The method ofincreasingthe therapeutically potent material available in raw liver, which consists in placing raw liver in an acid solution con-, ,taining anantiseptic and having I a p'l-I value within the limitspf pH 2 to pH 7.5, promoting autolysis by maintaining the temperature of the solution at ap loximately fli 0., and continuing the autolysis untilithe amino-acid content in reached a substz i.ntially con-. I

stant level.

7. The method of increasing the therapeutically potent material contained in raw liver by at least 7 2,5 times, which consists in mincing'raw liver by v 'in an aqueous solution of hydrochloric acid and:

I grinding of otherwise, placing the minced liver'. be adapted for taking .by-

an antiseptic in theproportions of approximately 200 grams of liver, '1,000 cc. water, 2 cc. concentrated hydrochloric acid, and 5 cc. of an antiseptic, such as chloroform, promoting active aurial irom liver without loss of the effective anti.

anemic properties containedjn the original liver from which the material is made, which consists in placing raw liver in an acid solution containing an antiseptic and having a pH value within the limits of pH 2 to pH 7.5, promoting autolysis of such liver by maintaining the solution at approximately 37 C., and continuing the autolysis until the amino-acid content 01 the solution has 7 reached a substantially constant level.

, 9. A method of increasing the yield 0! antianemic material which can be derived from raw liver which consists in placing raw liver in an acid solution containing an antiseptic and having a pH value within the limits of pH 2 to pH 7.5, promoting autolysis of such liver by maintaining the solution at approximately 37? C., and continu-f ing the autolysis until the amino-acid content of the solution has reached'a substantially constant level. a

10. An anti-anemic preparation containing at least as much of the anti-anemic properties as was contained in the original liver from which it was made, comprising a concentrate derived from liver autolyzed in a solution having a pH favorable for active autolysis and for such a length or time that the amino-acid content has reached a substantially constant level.

WILLIAM S. McEILROY. v WILLIAM F. HERRON. 

